Network analysis of the focal adhesion to invadopodia transition identifies a PI3K-PKCα invasive signaling axis

Abstract

In cancer, deregulated signaling can produce an invasive cellular phenotype. We modeled the invasive transition as a theoretical switch between two cytoskeletal structures: focal adhesions and extracellular matrix-degrading invadopodia. We constructed molecular interaction networks of each structure and identified upstream regulatory hubs through computational analyses. We compared these regulatory hubs to the status of signaling components from head and neck carcinomas, which led us to analyze phosphatidylinositol 3-kinase (PI3K) and protein kinase C α (PKCα). Consistent with previous studies, PI3K activity promoted both the formation and the activity of invadopodia. We found that PI3K induction of invadopodia was increased by overexpression of SH2 (Src homology 2) domain-containing inositol 5'-phosphatase 2 (SHIP2), which converts the phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] that is produced by PI3K activity to phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)], which is believed to promote invadopodia formation. Knockdown of PKCα had divergent effects on invadopodia formation, depending on the status of PI3K. Loss of PKCα inhibited invadopodia formation in cells with wild-type PI3K pathway status. Conversely, in cells with constitutively active PI3K (through activating PI3K mutants or lacking the endogenous opposing enzyme PTEN), PKCα knockdown increased invadopodia formation. Mechanistic studies revealed a negative feedback loop from PKCα that dampened PI3K activity and invasive behavior in cells with genetic hyperactivation of the PI3K pathway. These studies demonstrated the potential of network modeling as a discovery tool and identified PI3K and PKCα as interacting regulators of invasive behavior.

Fig 1. Src is the central hub of the invadopodia network

Protein components were included in the literature-based invadopodia network if they have been detected in invadopodia puncta by immunofluorescence. Node coloration indicates degree of connectedness to other nodes with red indicating the highest degree of connectedness and blue indicating the lowest degree of connectedness.

Fig 2. Centralities analysis of expanded networks

Using the Centiscape plugin for Cytoscape and R, the top ten proteins were ranked according to centrality measurements: degree (the number of binding partners), betweenness (the proportion of all shortest paths between 2 nodes going through a given node), stress (the number of all of the shortest paths within the network going through a given node), and sum of shortest paths (SoSP, node with the smallest sum of the shortest paths initiated to all other nodes in the network).

Fig 3. PI3K and PKCα analytes are included in a cluster of HPV-negative HNSCC associated with tumor recurrence

Heatmap of analytes selected for correlation with “recurred” or “nonrecurred” status were subjected to unsupervised clustering. Red color indicates high and blue coloring indicates low abundance in comparison to the median for that analyte. A large box outlines the “recurrence cluster” and asterisks mark potential invadopodia-associated analytes.

Fig 4. Inhibition of PI3K leads to a decrease in invadopodia and central focal adhesion number

A. Images of SCC61 HNSCC cells treated with diluent control (“DMSO”) or 25 μM LY294002. Arrowheads point to example invadopodia. B. Quantitation of invadopodia-mediated ECM degradation area/cell area, number of invadopodia per cell, and numbers of central or peripheral focal adhesion per cell. n≥110 cells per cell line from 3 independent experiments. Scale bar = 20 μm. *p<0.05; **p<0.01; ***p<0.001.

Fig 5. PKCα inhibition leads to divergent invadopodia phenotypes, depending on PI3K mutant status

A. Merged images of invadopodia (red; actin) and focal adhesions (green; vinculin) from SCC25 cells expressing empty vector (Control), or PI3K mutants (E545K, H1047R) with or without knockdown of PKCα (PKCα siRNA). Arrows point to focal adhesion belts surrounding invadopodia clusters. Matching fibronectin images are in Fig S3. B. Analyses of invadopodia and focal adhesion. n≥50 cells per cell line from 3 independent experiments. Scale bar = 20 μm. *p<0.05; **p<0.01; ***p<0.001.

Fig 6. PI metabolism promotes switching between cytoskeletal structures

A. Pie chart diagram of network analysis indicating the interaction of focal adhesion (green), or invadopodia (blue) molecules with selected phosphatidylinositol species (PIs). B. Fisher’s exact test of node enrichment in the neighborhood of PI species indicates that PI(3,4,5)P₃, and PI(3,4)P₂ are significantly associated with both invadopodia and focal adhesion molecules, whereas the precursor PI(4,5)P₂ is associated only with focal adhesions. PI(3,5)P₂ is significantly associated with invadopodia. Depth of connections = 2 for analysis shown (PI binding partners and first neighbors). C. Schematic of conversion between key PI species. Red indicates enzymes likely to promote PI(3,4)P₂ formation in epithelial cells that we tested. D. Images of SCC61 cells expressing vector only (control) or SHIP2-FLAG (SHIP2), or SHIP2-FLAG cells treated with 12.5 or 25 μM LY294002 (SHIP2+LY12.5 or LY25). E. Western blot of control and SHIP2-FLAG-expressing cells probed with an anti-Flag antibody. * indicates non-specific band detected by anti-FLAG antibody. Tubulin is the loading control. F. Invadopodia and focal adhesion analyses. n≥70 cells per cell line from 3 independent experiments. Scale bar = 20 μm. *p<0.05; **p<0.01; ***p<0.001.

Fig 7. PKCα negatively regulates both invadopodia formation and stability in PI3K mutant-expressing cells

A,B. SCC25 cells stably expressing H1047R PI3K and m1Venus-zyxin were transfected with tdTomato-F-tractin and control or PKCα siRNA. They were replated on FN-coated gelatin plates and serum- and growth factor-starved before stimulation with invadopodia media and live imaging. Frames were captured every 90 sec for 90 min. A. Images from example movies. Scale bars = 10 μm. B. Quantitation of the number of new invadopodia formed per cell binned by time after stimulation. n≥23 cells per cell line from 5 independent experiments. ***p<0.001 compared with control. C. SCC25 cells stably expressing tdTomato-F-ractin were cultured in invadopodia media without previous starvation and live movies were obtained. Invadopodia lifetime was quantitated as the length of time an invadopodia persisted after its formation. n≥21 cells per cell line from 4 independent experiments.

Fig 8. PKCα inhibits cellular PI3K activity

A. SCC25 cells stably expressing H1047R PI3K were transfected with control or PKCα-targeting siRNA (siPKCα #1 and #2). Control + LY, control cells treated with the PI3K inhibitor LY294002. Cells were fixed and stained with probes for PI(3,4,5)P₃ (red in Merge) and PI(4,5)P₂ (green in Merge). Images on right (“Ratio”) have been color converted to show the ratio of PI(3,4,5)P₃ to PI(4,5)P₂ and have a corresponding ratiometric color scale bar. B. Quantification of (PI(3,4,5)P₃/PI(4,5)P₂) fluorescence intensity ratios in individual cells from images. n≥15 cells per condition from 3 independent experiments. C. Representative Western blots showing abundance of PKCα; tubulin loading control; pSer⁴⁷³-Akt (pAkt) and total Akt (Akt) in H1047R control and PKCα-siRNA cells. D. Quantification of Active-Akt (pAkt/Total Akt) from n=3 Western blots. Mean+/− standard error plotted. E. Western blot of control, p85 wild type-FLAG-(p85 -WT), and p85 S361/652A-FLAG (p85-SA)-expressing SCC25-H1047R cells probed with an anti-Flag antibody. Tubulin loading control. F. Invadopodia-associated ECM degradation in p85-overexpressing cells. n≥62 cells per cell line from 3 independent experiments. Scale bar = 20 μm. * p<0.05, *** p<0.001.